Topography of pleural epithelial structure enabled by en face isolation and machine learning

Pleural epithelial adaptations to mechanical stress are relevant to both normal lung function and parenchymal lung diseases. Assessing regional differences in mechanical stress, however, has been complicated by the nonlinear stress–strain properties of the lung and the large displacements with ventilation. Moreover, there is no reliable method of isolating pleural epithelium for structural studies. To define the topographic variation in pleural structure, we developed a method of en face harvest of murine pleural epithelium. Silver-stain was used to highlight cell borders and facilitate imaging with light microscopy. Machine learning and watershed segmentation were used to define the cell area and cell perimeter of the isolated pleural epithelial cells. In the deflated lung at residual volume, the pleural epithelial cells were significantly larger in the apex (624 ± 247 μm²) than in basilar regions of the lung (471 ± 119 μm²) (p < 0.001). The distortion of apical epithelial cells was consistent with a vertical gradient of pleural pressures. To assess epithelial changes with inflation, the pleura was studied at total lung capacity. The average epithelial cell area increased 57% and the average perimeter increased 27% between residual volume and total lung capacity. The increase in lung volume was less than half the percent change predicted by uniform or isotropic expansion of the lung. We conclude that the structured analysis of pleural epithelial cells complements studies of pulmonary microstructure and provides useful insights into the regional distribution of mechanical stresses in the lung.     

Regional deposition and retention of particles in shallow, inhaled boluses: effect of lung volume

The regionaldeposition of particles in boluses delivered to shallow lung depths andtheir subsequent retention in the airways may depend on the lung volumeat which the boluses are delivered. To evaluate the effectof end-inspiratory lung volume on aerosol bolus delivery, we hadhealthy subjects inhale radiolabeled, monodisperse aerosol(^99mTc-iron oxide, 3.5-µm massmedian aerodynamic diameter) boluses (40 ml) to a volumetric frontdepth of 70 ml into the lung at lung volumes of 50, 70, and 85% oftotal lung capacity (TLC) end inhalation. By gamma camera analysis, wefound significantly greater deposition in the left (L) vs. right (R)lungs at the 70 and 85% TLC end inhalation; ratio of deposition in Lto R lung, normalized to L-to-R ratio of lung volume (mean L/R), was1.60 ± 0.45 (SD) and 1.96 ± 0.72, respectively(P < 0.001 for comparison to 1.0) for posterior images. However, at 50% TLC, L/R was 1.23 ± 0.37, not significantly different from 1.0. These data suggest that the L andR lungs may be expanding nonuniformly at higher lung volumes. On theother hand, subsequent retention of deposited particles at 2 and 24 hpostdeposition was independent of L/R at the various lung volumes. Thusasymmetric bolus ventilation for these very shallow boluses does notlead to significant increases in peripheral alveolar deposition. Thesedata may prove useful for 1)designing aerosol delivery techniques to target bronchial airways and2) understanding airway retention ofinhaled particles.

     

Screening for improved cell performance: selection of subclones with altered production kinetics or improved stability by cell sorting

One of the major problems in the biotechnology industry is the selection of cell lines well suited for production of biopharmaceutical proteins. Usually, the most important selection criterion is the cell specific production rate. Nevertheless, a good producer cell line should have a number of additional advantageous properties, which allow the cell line to perform well in the type of bioreactor chosen for the process. However, the time and work required to select for high production rates as well as the lack of methods to specifically select for other cellular properties, usually prevents researchers from including such criteria into their screening program.With the Single Cell Secretion Assay it is possible to measure the specific production rates of individual cells by catching secreted product in an artificial matrix applied to the cell surface. Flow cytometric cell sorting then allows selection of rare cells with high production rates, which occur at frequencies as low as 10(-6). By combining this method with culture conditions that bring out a desired cellular property, we were able to isolate subclones with similar production rates, but improved performance from a recombinant Chinese hamster ovary cell line producing a human monoclonal antibody. The two desired cellular properties screened for were a non-growth associated production kinetic and improved stability in the absence of selective pressure.     

Requirement for the p75 TNF-alpha receptor 2 in the regulation of airway hyperresponsiveness by gamma delta T cells

In a recent study, we found that TNF-alpha negatively regulates airway responsiveness through the activation of gammadelta T cells. The biological activities of TNF-alpha are mediated by two structurally related but functionally distinct receptors, p55 (TNFR1) and p75 (TNFR2), which are independently expressed on the cell surface. However, the relative importance of either TNFR in airway hyperresponsiveness (AHR) is unknown. To investigate the importance of these TNFRs in the development of allergen-induced AHR, p55-deficient and p75-deficient mice were sensitized to OVA by i.p. injection and subsequently challenged with OVA via the airways; airway responsiveness to inhaled methacholine was monitored. p75-deficient mice developed AHR to a similar degree as control mice. In contrast, p55-deficient mice, which were sensitized and challenged with OVA, failed to develop AHR. In p55-deficient mice, both the numbers of eosinophils and levels of IL-5 in bronchoalveolar lavage fluid were significantly lower than in sensitized/challenged control mice (p < 0.05). However, depletion of gammadelta T cells resulted in significant increases in AHR in the p55-deficient mice, whereas no significant effect of gammadelta T cell depletion was evident in the p75-deficient mice. These data indicate that, in the absence of TNFR1 (p55), where TNF-alpha uses the p75 pathway exclusively, the development of AHR is regulated by gammadelta T cells.     

Cloning and analysis of the mating type genes from Cochliobolus heterostrophus

Cochliobolus heterostrophus, a heterothallic Ascomycete, has a single mating type locus with two alternate forms called MAT-1 and MAT-2. MAT-1 was cloned by complementing a MAT-2 strain using a cosmid library from a MAT-1 strain and screening for a homothallic transformant. The cosmid recovered from this transformant was able to re-transform a MAT-2 strain to homothallism and MAT identity was proven by restriction fragment length polymorphism and conventional genetic mapping. All homothallic transformants could mate with either MAT-1 or MAT-2 strains, although the number of ascospores produced by self matings or crosses to MAT-2 strains was low. Progeny of selfed homothallic transformants were themselves homothallic. MAT-2 was cloned by probing a cosmid library from a MAT-2 strain with a fragment of insert DNA from the MAT-1 cosmid. A 1.5 kb subclone of either MAT-containing cosmid was sufficient to confer mating function in transformants. Examination of the DNA sequence of these subclones revealed that MAT-1 and MAT-2 contain 1297 by and 1171 bp, respectively, of completely dissimilar DNA flanked by DNA common to both mating types. Putative introns were found (one in each MAT gene) which, when spliced out, would yield open reading frames (ORFs) that occupied approximately 90% of the dissimilar DNA sequences. Translation of the MAT-1 ORF revealed similarity to the Neurospora crassa MATA, Podospora anserina mat–, and Saccharomyces cerevisiae MAT1 proteins; translation of the MAT-2 ORF revealed similarity to the N. crassa MATa, P. anserina mat+, and Schizosaccharomyces pombe mat-Mc proteins. These gene products are all proven or proposed DNA binding proteins. Those with similarity to MAT-2 are members of the high mobility group.The first three authors contributed equally to the work     

The significance of leaf pubescence for the absorption of global radiation by Tussilago farfara L.

Summary The spectral properties of juvenile and adult leaves of Tussilago farfara L. were measured for the wave range from 400 to 1350 nm. The significance of leaf pubescence for the absorption of global radiation was determined. The absorption is lowered by epidermal harrs only for the visible part of the spectrum (400–1350 nm). Absorptivities of the infrared do not vary whether hairs are present or not. The spectral properties of the upper and the lower surfaces without hairs are not equal. The hairs on the lower surface increase the amount of infrared radiation that is reflected by the upper surface. As shown by calculations of the absorbed radiation, pubescence has little influence on energy input. If leaves are put in an inverse position, the absorption of the global radiation energy is lowered by 15% compared with the same leaf in the regular position.     

Zur Biologie, Taxonomie und Chronologie der Conodonten

The actual state of knowledge concerning the conodont animal is critically reviewed. All soft tissue documented by the specimens of the conodont animal were interpreted by the British collectors as Craniota (Vertebrata) features. Opinions on the precise affinities, however, remain controverse: The alleged chorda could have been a gut, bilateral symmetry, myomery, apatitic biomineralisation occur also in Invertebrata, caudal fins do in Chaetognatha. Even the lobes in the capital area interpreted as sensory organs could have served as aiding organs for food intake. Therefore, the originally sensational findings are reduced to the core issue that the conodont animal had an eel-like body and at the blunt end of it, behind peculiar lobes, the mineralized conodont elements were arranged. As a consequence, a relationship to the Protochordata cannot be excluded. A systematic position of the conodont animal as possible Vertebrata is likely only if organized below the Ostracodermata which is particularly so because conodonts occur so early in the Cambrian. In the second part of the article conodonts are discussed as guide fossils. The comprehensive, empirically grown taxonomy of the single elements is the prerequisite for recognizing outstanding, high resolution stratigraphy. In comparison, a multielement taxonomy remains as long problematical, as it is based on assumed, mostly statistically reconstructed rather than on natural multielement apparatuses. Taxonomical and nomenclatorial operations built on assumed apparatus affinity can bear heavily on nomenclature and stratigraphy of platform elements. It can be shown at the example of the Devonian that the platform elements have provided particularly in the pelagic facies a Standard Conodont Zonation that covers time almost without any gaps. The basis for this is that platform single element species and their phylogenetic development are extra-well known. In a special paragraph, history and applicability of the Standard Zonation is discussed in details. Different conodont biofacies are developed between the pelagic realm where the Standard Zonation originated, and the coastal areas so that conodonts can indicate temporal and bathymetric relationships of the mother rocks. In addition alternative conodont zonations were introduced mainly for the shallow water. Because of their conspicuous stratigraphic significance in the Devonian, conodonts were employed by International Committees to redefine Series and Stage Boundaries. For the boundary selection species were utilized from phylognetic lineages as only from tight evolution their point of origination could be well identified and recognized in worldwide correlation. The GSSPs (Global Stratotype Section and Point) within the Devonian are discussed in detail. In recent times, attempts are made at calibrating the duration of conodont zones on the basis of their well known rate of evolution and high temporal resolution. These analyses have to be continued in order to demonstrate the absolute time relationship of condont zones in more and continuous detail. Currently the phyletically controlled Standard Conodont Zonation is the authoritative stratigraphic method in the Devonian. It has provided together with the GSSPs precise and valuable fix points for a Global Time Scale (GTS). In combination with additional stratigraphie methods the Standard Conodont Zonation may provide further refinements in the future.     

Different potentials of gamma delta T cell subsets in regulating airway responsiveness: V gamma 1+ cells, but not V gamma 4+ cells, promote airway hyperreactivity, Th2 cytokines, and airway inflammation

Allergic airway inflammation and hyperreactivity are modulated by gammadelta T cells, but different experimental parameters can influence the effects observed. For example, in sensitized C57BL/6 and BALB/c mice, transient depletion of all TCR-delta(+) cells just before airway challenge resulted in airway hyperresponsiveness (AHR), but caused hyporesponsiveness when initiated before i.p. sensitization. Vgamma4(+) gammadelta T cells strongly suppressed AHR; their depletion relieved suppression when initiated before challenge, but not before sensitization, and they suppressed AHR when transferred before challenge into sensitized TCR-Vgamma4(-/-)/6(-/-) mice. In contrast, Vgamma1(+) gammadelta T cells enhanced AHR and airway inflammation. In normal mice (C57BL/6 and BALB/c), enhancement of AHR was abrogated only when these cells were depleted before sensitization, but not before challenge, and with regard to airway inflammation, this effect was limited to C57BL/6 mice. However, Vgamma1(+) gammadelta T cells enhanced AHR when transferred before challenge into sensitized B6.TCR-delta(-/-) mice. In this study Vgamma1(+) cells also increased levels of Th2 cytokines in the airways and, to a lesser extent, lung eosinophil numbers. Thus, Vgamma4(+) cells suppress AHR, and Vgamma1(+) cells enhance AHR and airway inflammation under defined experimental conditions. These findings show how gammadelta T cells can be both inhibitors and enhancers of AHR and airway inflammation, and they provide further support for the hypothesis that TCR expression and function cosegregate in gammadelta T cells.